The applicant will study virulence determinants in members of the Mycobacterium avium complex (MAC) which re pathogens which can infect immunocompromised patients. MAC organisms cause the most common bacterial infection encountered in advanced AIDS. MAC and other mycobacteria (including those that cause human tuberculosis) have been very difficult to study because of their slow growth and the lack of easily-manipulated genetic systems. In addition, mycobacteria can grow within phagocytic cells, an environment which cannot be easily duplicated on laboratory media. Because of these difficulties in working with mycobacteria different strategies are proposed to identify genes likely to play important roles in virulence. Genes which are required in pathogenesis are likely to be regulated. Genes expressed uniquely during infection of a macrophage- like cell line will be identified by subtractive hybridization. In addition, secreted proteins and those expressed on the surface are required for interaction with host cells are likely to be important in the establishment and maintenance of infection. A fusion vector will be constructed in Streptomyces (an organism which has similar codon usage and signal sequence structure to mycobacteria) which allows the screening of randomly-cloned MAC DNA for the presence of signal sequences which allow secretion of a Streptomyces protease. Identified genes will then be used to probe for expression within macrophages. Finally, workers in Dr. Mekalanos' laboratory have identified several infection-specific genes expressed by Salmonella, another intracellular pathogen. These will be used to search for homologous MAC sequences. Any genes identified by the above methods will be sequences and adjacent genes and regulatory sequences will be identified. Ultimately, the applicant hopes to disrupt candidate virulence genes by homologous recombination using a strategy which has been successful in Mycobacterium smegmatis.